We have previously found that docosahexaenoic acid (DHA, 22:6n-3), a highly polyunsaturated n-3 fatty acid, promotes the accumulation of phosphatidylserine (PS) and prevents apoptotic cell death in a PS- and PI3 kinase-dependent manner in neuronal cells. We have also demonstrated that n-3 fatty acid deficiency or chronic ethanol exposure markedly decreased the PS content specifically in neuronal cells where DHA is highly enriched. We have established that docosapentaenoic acid (DPA, 22:5n-6), which replaces DHA during n-3 deficiency, is not as effective as DHA in accumulating PS or preventing apoptotic cell death, thus adversely affecting neuronal survival under n-3 deficiency conditions. Similarly, long term ethanol exposure during prenatal and developmental period depletes PS from neuronal membranes through the inhibition of PS biosynthetic activities. During this period, we continued our investigation of the signaling mechanisms underlying effects of DHA on cell survival. We found that PS levels were in direct correlation with the DHA content in neuronal cells. Although silencing or over-expressing PSS-1 and PSS-2 genes that are responsible for PS synthesis considerably altered their mRNA levels, no significant changes were observed for the protein or PS levels. Alternatively, incubating the cells directly with PS did not alter the PS content significantly, either, indicating that DHA uniquely modifies the PS pool in living cells. The protection against apoptotic cell death induced by serum starvation was sensitive to the PS level altered by the DHA status. The increase of PS resulted in faster translocation and phosphorylation of Akt upon IGF stimulation. The PI3 kinase activation was not affected, indicating that translocation of Akt is the target for the PS involvement in survival signaling pathways. It appeared that the ionic interaction between the acidic phospholipid PS and the basic residues (Arg, Lys) located away from the PIP3-binding pocket in the PH domain of Akt played an important role in faster translocation seen in DHA enriched cells, since the DHA effect was no longer observed with the inhibition of PS accumulation or mutations of these basic residues with neutral amino acid (Ala). DPA was less effective in accumulating PS, and accordingly less efficient in facilitating Akt translocation or preventing apoptosis. Consistently, in vivo reduction of DHA by dietary depletion of n-3 fatty acids decreased hippocampal PS and increased the susceptibility of hippocampal neurons to apoptosis in cultures. These data demonstrate a novel mechanism that membrane PS concentration introduced by DHA promotes a targeted event in PI3-kinase/Akt signaling, significantly impacting neuronal survival. It is suggested that cell survival inadequately supported by DPA may contribute to neurological deficits associated with n-3 fatty acid deficiency. For mechanistic understanding of our previous finding that DHA uniquely promotes hippocampal differentiation, the role of RXR for which DHA is an endogenous ligand is being investigated. At the same time, a method for evaluating neurite lengths semi-automatically in multiple samples is being established. As a part of our continuing efforts to investigate protein conformational changes due to molecular interaction, the interaction between human serum albumin (HSA) and fatty acids was examined using chemical cross-linking and mass spectrometry. Interaction of HSA with monounsaturated and polyunsaturated fatty acids including oleic acid (OA), arachidonic acid (AA) and docosahexaenoic acid (DHA) resulted in a local conformational change involving the side chain movement of K402 and K541 in domain III. The cross-linking data also indicated that side chains of K205 (IIA) and K466 (IIIA) moved closer toward each other upon binding with AA or DHA, but not with OA, suggesting that the conformation of HSA bound to mono- and polyuuunsaturated fatty acid is distinctively different. This approach is particularly useful in probing the solution structure of a protein in biological media and also can serve as complement to X-ray crystallography. In addition, progress has been made in studying non-covalent interactions at different stages of Ras protein activation. By using nano-electrospray ionization, the formation of Ras-GTP and Ras-GDP was detected. The subsequent interaction of GTP-bound Ras with Ras-binding-domain of Raf-1 (RBD) is now under investigation.